The phytohormone abscisic acid (ABA) is important for the regulation of abiotic stress responses (such as drought, salinity, cold shock, wounding, pathogen attack) and seed development and dormancy. Screens for mutants with altered abiotic stress responses or seed dormancy have been used frequently and resulted in the identification of genes important for ABA biosynthesis and ABA signal transduction. Via such a screen the Arabidopsis ABA-insensitive mutants abi1-1 and abi2-1 have been identified (Koornneef et al. 1984, Physiol Plantarum 61: 377-383).
Cloning and characterization of the AtABI1 gene revealed that it encodes for a serine/threonine protein phosphatase type 2C (PP2C, Leung et al. 1994, Science 264: 1448-1452; Meyer et al. 1994, Science 264: 1452-1455). AtABI2 also encodes for a protein phosphatase type 2C. abi1-1 and abi2-1 mutants carry mutations in the AtABI1 and AtABI2 genes, which result in identical Gly-to-Asp substitutions at equivalent positions (Leung et al. 1997, Plant Cell 9: 759-771). Both mutants were shown to have reduced phosphatase activity (Bertauche et al. 1996, Eur J Biochem 241: 193-200; Leung et al. 1997, supra), which would suggest that AtABI1 and AtABI2 are positive regulators of ABA sensitivity. However, constitutive over-expression of AtABI1 inhibited ABA action in maize protoplast, and reduction-of-function mutants of AtABI1 and AtABI2 were shown to have hypersensitive responses to ABA (Scheen 1998, PNAS 95:975-980; Gosti et al. 1999, Plant Cell 11: 1897-1909; Merlot et al. 2001, Plant J 25:295-303). Altogether, it was therefore concluded that AtABI1 and AtABI2 are negative regulators of the ABA response. The exact mechanism by which the mutations in abi1-1 and abi2-1 induce ABA insensitivity is still unknown, although it might be related to the preferential nuclear localization of the mutated proteins (Moes et al. 2008, Plant J 54: 806-819). AtABI1 and AtABI2 are important for seed dormancy but also for seedling growth and regulation of stomatal aperture, suggesting that these proteins act before major branch points that control tissue-specific ABA signaling cascades (Leung et al. 1997, supra).
Seventy-six PP2Cs have been identified in Arabidopsis, of which one subgroup subgroup PP2C-A), consisting of nine genes, has been associated with ABA signal transduction (Schweighofer et al. 2004, Trends in Plant Science Vol 9: 236-243). Several of the Arabidopsis genes belonging to this group were also found to encode for negative regulators of the ABA response. For example AtP2C-HA (Rodriguez et al. 1998, Plant Mol Biol 38: 879-883)(also named AtHAB1, Saez et al. 2004, Plant J 37: 354-369) is a repressor of the ABA signalling pathway that regulates numerous ABA responses such as stomatal closure, seed germination and inhibition of vegetative growth. Also HAB2 seems to have a similar regulatory role. AtPP2CA (Kuhn et al. 2006, Plant Physiol 140:127-139) has also been described as being a negative regulator of ABA, with the mutant showing ABA hypersensitivity. Interestingly, the gene disruption mutant showed an ABA hypersensitive stomatal closure response, while transpiration (water loss) of the mutant was no different than in wild type plants. Also Yoshida et al. (2006, Plant Physiology 140: 115-126) studied a missense loss-of-function mutation in AtPP2CA, which had 1/100th protein phosphatase activity of the wild type, and whereby the mutant Arabidopsis plants showed ABA hypersensitivity during seed germination, but mutant plants did not show any change in drought tolerance compared to wild type (page 124, LH Column, last paragraph).
ABI1 and ABI2 are repressors of ABA signalling pathways that regulate many ABA responses, such as stomatal closure, osmotic water permeability of plasma membranes, drought-induced resistance and rhizogenesis, response to glucose, high light stress, seed germination and inhibition of vegetative growth. AHG1 (At5g51760) is a negative regulator of ABA during seed germination (Nishimura et al. 2007, Plant J 50: 935-949) and AHG3 (At3g11410) is a negative regulator of ABA during seed germination and cold acclimation. Three other, At5g9220, At2g29380 and At1g07430, may after all not be involved in ABA signalling as null mutations did not reveal any change in sensitivity to ABA (Yoshida et al, 2006: Plant Physiology 140:115-126).
ABA biosynthesis and signalling is, thus, extremely complex, and although various genes have been identified in Arabidopsis which appear to be involved (group PP2C-A), their role in ABA dependent responses, such as abiotic stress, seed dormancy and seedling growth is to a large extent unclear. In addition, protein sequences show little conservation and amino acid sequences share little sequence identity. The genes are grouped together phylogenetically, based on protein domains (or motifs) such as the catalytic domain (protein serine/threonine phosphatase like domain) which is typically located at the C-terminal of the proteins. The N-terminal varies considerably and may play a role in substrate binding or provide specific attachment sites to signalling complexes. In addition to the in vivo function being unclear, also little is known about the subcellular localization, substrates and specificity of the enzymes or how these monomeric enzymes are regulated in vivo.
WO2007/088234 describes that combined inactivation of ABI1 and HAB1 strengthens the response to ABA leads to Arabidopsis plants which are resistant to salinity and hydric stress. Tomato orthologs of ABI1 (SGN-U231558) and HAB1 (SGN-U217609) are presented in FIGS. 6 and 7. See also Saez et al. 2006, Plant Physiol 141:1389-1399.
Although abi1 hab1 double mutants lead to drought tolerance in Arabidopsis thaliana, there remains a need for providing genes which are suitable for generating drought tolerance in crop plants, especially in field crops (e.g. rice, maize, soybean, wheat, barley, rye, sorghum, Brassica, etc.) and vegetable crops (e.g. tomato, cucumber, onion, carrot, cabbage, cauliflower, broccoli, watermelon, melon, lettuce, leek, spinach, radish, potato, artichoke, corn salad, pumpkin, squash, bean, peas, pepper, etc.). Especially in vegetable crops water shortage can be a big problem, as roots of many crops are shallow and as products are often sold fresh and water shortage can lead to reduced vegetable quality and reduced yield. Fruit and seed vegetables, such as tomatoes, are sensitive to water shortage during flowering and during fruit- and seed development. Fruit-set can be seriously reduced by water shortage during fruit development. Common practice to deal with potential water stress situations is to irrigate crops and/or to plant cultivars or varieties with drought tolerance, in as far as these are available. Also mulches and row covers may be used.
Despite breeding efforts, tomato plants remain sensitive to drought and no commercial cultivar with drought tolerance is available.
The present invention provides new genes, referred to as SlPP2C1, suitable for generating drought tolerant crop plants, especially tomato plants and other vegetable plants. The present invention also provides methods of generating drought tolerant plants. Also provided are the drought tolerant plants, seeds and plant parts (harvested fruit, etc.) themselves.